There is growing evidence that the development of insulin resistance is preceded and precipitated by alterations in the partitioning of fat between the adipose tissue and the muscle, liver and pancreas. Intracellular accumulations of triglyceride and fatty acid metabolites lead to acquired insulin signaling defects and insulin resistance. Importantly, the skeletal muscle tissue of offspring of diabetics has been shown to be morphologically (smaller, less dense mitochondria) and functionally (low oxidative capacity) abnormal. One can therefore hypothesize that the inability of the adipose organ to expand to accommodate excess calories (impaired adipogenesis) results in adipose tissue hypertrophy, ectopic fat deposition and insulin resistance in muscle, liver and adipose in predisposed subjects. Our hypothesis is clearly supported by the following data: 1. Pima Indians with larger abdominal fat cells are more likely to develop insulin resistance and T2DM. 2. TZDs improve insulin sensitivity partially by inducing adipoctye differentiation. 3. Subjects with acquired total lipodystrophy are severely diabetic. 4. Insulin sensitivity is inversely related to triglycerides content of muscle and its mitochondrial content. We have designed the proposed studies to test whether an 8-week 40% overfeeding regime will significantly increase ectopic fat deposition, insulin resistance and decrease muscle oxidative capacity in 20 individuals with hypertrophic adipocytes more than in 20 individuals with hyperplasic adipoctyes matched for total adiposity. Our hypothesis is that individuals with larger adipocytes have impaired differentiation of preadipocytes, muscle cells with impaired oxidative capacity and insulin resistance, increased ectopic fat content and whole body insulin resistance. We also hypothesize that subjects with hypertrophic adipocytes will be more vulnerable to fat gain with overfeeding than subjects with hyperplasic adipocytes. These studies will be conducted by investigating the molecular and morphological characteristics of subcutaneous adipose and skeletal muscle tissues and primary cultures of skeletal muscle and adipose-derived stroma. In addition we will measure in situ ectopic fat (liver and muscle by MRS), in vivo insulin sensitivity (liver, muscle and adipose by a clamp) and in vivo metabolic flexibility (clamp and respiratory chamber).The proposed studies have been designed to investigate the respective roles of the adipose and skeletal muscle tissues in the development of insulin resistance in response to overfeeding. We hypothesize that individuals with both a poor potential for adipose tissue expansion and an impaired function of muscle mitochondria will have the most deterioration of insulin sensitivity after 8 weeks of overfeeding. [unreadable] [unreadable] [unreadable] [unreadable]